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| 沙库巴曲/缬沙坦对心力衰竭兔心肌细胞的保护作用及机制研究 |
| 庄金龙1,2,钱涛铭1,林庚海2,陈华2,阮发晖2,林惠萍2,刘莉1,3* |
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(1. 黑龙江中医药大学, 哈尔滨 150040;
2. 厦门大学附属东南医院心血管内科, 漳州 363000;
3. 黑龙江中医药大学附属第一医院心血管病一科, 哈尔滨 150040
*通信作者) |
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| 摘要: |
| 目的 探讨沙库巴曲（Sac）/缬沙坦（Val）对多柔比星（DOX）诱导的心力衰竭兔心肌细胞的保护作用及机制。方法 选取30只新西兰兔建立DOX诱导的心力衰竭兔模型，25只造模成功，将造模成功的实验兔随机分为模型组（DOX组，9只）、Val干预组（DOX＋Val组，8只）、Sac/Val干预组（DOX＋Sac/Val组，8只）；另取8只新西兰兔作为空白组。DOX＋Val组每次灌胃Val混悬液4.65 mg/kg，DOX＋Sac/Val组每次灌胃Sac/Val混悬液9.3 mg/kg，空白组及DOX组每次灌胃等体积的蒸馏水；各组均每日灌胃2次，连续8周。给药8周后，用超声心动图检测兔左心室舒张末期内径（LVDD）、左心室收缩末期内径（LVSD）、左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）；计算全心重量指数（HMI）及左心室重量指数（LVMI）；通过H-E和马松染色观察心肌组织病理形态及纤维化情况；通过透射电镜观察心肌细胞超微结构；采用TUNEL检测观察心肌细胞凋亡情况，并计算心肌细胞凋亡率；采用ELISA法检测血清氨基末端脑利尿钠肽前体（NT-proBNP）、超敏肌钙蛋白I（Hs-cTNI）、血管紧张素Ⅱ（Ang Ⅱ）、醛固酮（ALD）、心房利尿钠肽（ANP）、脑利尿钠肽（BNP）、环磷酸鸟苷（cGMP）、蛋白激酶G（PKG）水平；采用qPCR检测心肌组织利尿钠肽受体A（NPR-A）、cGMP特异性磷酸二酯酶5A［PDE5A（cGMP）］、PKG、Bcl-2、Bax、caspase 3 mRNA 的表达；采用蛋白质印迹法检测心肌组织磷酸化cAMP反应元件结合蛋白（p-CREB）、磷酸化Bcl-2相关死亡促进因子（p-Bad）、Bcl-2、Bax、caspase 3蛋白的表达。结果 与空白组相比，DOX组兔的LVDD、LVSD均增大（均P＜0.01），LVEF、LVFS均降低（均P＜0.01），HMI、LVMI均升高（均P＜0.01）；心肌细胞凋亡增加，心肌细胞凋亡率升高（P＜0.01）；NT-proBNP、Hs-cTNI、Ang Ⅱ、ALD、ANP、BNP、cGMP、PKG水平和NPR-A、PDE5A（cGMP）、PKG、p-CREB、Bax、caspase 3表达均升高（均P＜0.01），Bcl-2表达降低（P＜0.01），p-Bad表达差异无统计学意义（P＞0.05）。与DOX组相比，DOX＋Sac/Val组和DOX＋Val组的LVDD、LVSD均减小（均P＜0.01），LVEF、LVFS均升高（均P＜0.01），HMI、LVMI均降低（均P＜0.01）；心肌细胞凋亡减少，心肌细胞凋亡率降低（P＜0.01）；NT-proBNP、Hs-cTNI、Ang Ⅱ、ALD、ANP、BNP、cGMP、PKG水平和NPR-A、PDE5A（cGMP）、PKG、Bax、caspase 3表达均降低（均P＜0.01），Bcl-2表达均升高（均P＜0.01）；DOX＋Sac/Val组的p-CREB、p-Bad表达均升高（均P＜0.01），但DOX＋Val组p-CREB、p-Bad无明显变化（均P＞0.05）。与DOX＋Val组相比，DOX＋Sac/Val组上述指标中除LVEF、LVFS、NPR-A、ANP、BNP、cGMP、PDE5A（cGMP）、PKG、p-CREB、p-Bad、Bcl-2均升高外，其余指标均降低（均P＜0.05）。心肌组织病理学和透射电镜结果显示Sac/Val可有效保护心肌细胞、减少细胞凋亡、减轻心肌纤维化，且该效果明显优于Val。结论 Sac/Val可有效减少心力衰竭兔心肌细胞凋亡，改善心脏功能及心肌纤维化，且该效果优于Val，其作用机制可能与活化NPR-A/cGMP/PKG信号通路和抑制肾素-血管紧张素-醛固酮系统有关。 |
| 关键词: 沙库巴曲 缬沙坦 心力衰竭 心肌细胞 细胞凋亡 心功能 |
| DOI：10.16781/j.CN31-2187/R.20240761 |
| 投稿时间：2024-11-10修订日期：2025-01-03 |
| 基金项目:国家自然科学基金（82074346），福建省自然科学基金（2023J011835），漳州市自然科学基金（ZZ2018J14）. |
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| Protective effects and mechanism of sacubitril/valsartan on cardiomyocytes of rabbits with heart failure |
| ZHUANG Jinlong1,2,QIAN Taoming1,LIN Genghai2,CHEN Hua2,RUAN Fahui2,LIN Huiping2,LIU Li1,3* |
(1. Heilongjiang University of Chinese Medicine, Harbin 150040, Heilongjiang, China;
2. Department of Cardiovasology, Southeast Hospital Affiliated to Xiamen University, Zhangzhou 363000, Fujian, China;
3. Department of Cardiovasology (Ⅰ), The First Affiliated Hospital, Heilongjiang University of Traditional Chinese Medicine, Harbin 150040, Heilongjiang, China
*Corresponding author) |
| Abstract: |
| Objective To study the protective effects and mechanism of sacubitril (Sac)/valsartan (Val) on cardiomyocytes of rabbits with heart failure induced by doxorubicin (DOX). Methods Thirty New Zealand rabbits were selected to establish DOX-induced heart failure rabbit model. Twenty-five rabbits with successful modeling were randomly assigned to model group (DOX group, n＝9), DOX＋Val group (n＝8), and DOX＋Sac/Val group (n＝8); and another 8 New Zealand rabbits were selected as blank group. The DOX＋Val group was gavaged with 4.65 mg/kg Val suspension each time, the DOX＋Sac/Val group was gavaged with 9.3 mg/kg Sac/Val suspension each time, and the blank group and DOX group were gavaged with equal volume of distilled water each time. Each group was gavaged twice a day for 8 weeks. After 8 weeks of administration, echocardiography was used to measure left ventricular end-diastolic diameter (LVDD), left ventricular end-systolic diameter (LVSD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS). The heart mass index (HMI) and left ventricular mass index (LVMI) were calculated. The pathological morphology and myocardial fibrosis of myocardial tissue were observed by hematoxylin-eosin (H-E) and Masson staining. The ultrastructure of cardiomyocytes was observed by transmission electron microscope. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to observe cardiomyocytes apoptosis and apoptosis rate was calculated. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), high-sensitivity cardiac troponin I (Hs-cTNI), angiotensinⅡ (AngⅡ), aldosterone (ALD), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG) in serum. Quantitative polymerase chain reaction (qPCR) was used to detect the expression of natriuretic peptide receptor A (NPR-A), cGMP-specific phosphodiesterase 5A (PDE5A［cGMP］), PKG, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), and cysteine aspartate protease 3 (caspase 3) mRNA in myocardial tissue. Western blotting was used to detect the expression of phosphorylated cAMP response element-binding protein (p-CREB), phosphorylated Bcl-2 related death promoting factor (p-Bad), Bcl-2, Bax, and caspase 3 proteins in myocardial tissue. Results Compared with the blank group, the LVDD and LVSD in the DOX group were increased (both P＜0.01), the LVEF and LVFS were decreased (both P＜0.01) and the HMI and LVMI were increased (both P＜0.01); the apoptosis and apoptosis rate of cardiomyocytes were increased (P＜0.01); the levels of NT-proBNP, Hs-cTNI, AngⅡ, ALD, ANP, BNP, cGMP and PKG and the expression of NPR-A, PDE5A (cGMP), PKG, p-CREB, Bax and caspase 3 were all increased (all P＜0.01), while the expression of Bcl-2 was decreased (P＜0.01), and the expression of p-Bad had no significant difference (P＞0.05). Compared with the DOX group, the LVDD and LVSD of the DOX＋Sac/Val group and DOX＋Val group were decreased (all P＜0.01), the LVEF and LVFS were increased (all P＜0.01) and the HMI and the LVMI were decreased (all P＜0.01); the apoptosis and apoptosis rate of cardiomyocytes were decreased (all P＜0.01); the levels of NT-proBNP, Hs-cTNI, Ang Ⅱ, ALD, ANP, BNP, cGMP and PKG and the expression of NPR-A, PDE5A (cGMP), PKG, Bax and caspase 3 were all decreased (all P＜0.01), while the expression of Bcl-2 was increased (P＜0.01); and the expression of p-CREB and p-Bad was increased in the DOX＋Sac/Val group (both P＜0.01), but there was no significant difference in the DOX＋Val group (both P＞0.05). Compared with the DOX＋Val group, the DOX＋Sac/Val group showed a decrease in all indicators except for LVEF, LVFS, NPR-A, ANP, BNP, cGMP, PDE5A (cGMP), PKG, p-CREB, p-Bad, and Bcl-2, which were all elevated (all P＜0.05). Myocardial pathology and transmission electron microscopy showed that Sac/Val effectively protected cardiomyocytes, reduced cardiomyocytes apoptosis and myocardial fibrosis, and these effects were significantly better than those of Val. Conclusion Sac/Val can effectively reduce cardiomyocytes apoptosis, improve cardiac function and reduce myocardial fibrosis in rabbits with heart failure, and these effects are superior to Val. Its mechanism may be related to activating the NPR-A/cGMP/PKG signaling pathway and inhibiting renin-angiotensin-aldosterone system. |
| Key words: sacubitril valsartan heart failure cardiomyocytes apoptosis cardiac function |
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